The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.
Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.
Mosquitoes are the deadliest organisms on earth, claiming the lives of more than one million people per year and place more than half the world population at risk of exposure to the pathogens they transmit, while biting1. These insects rely on a wide range of cues (i.e., thermal, visual, mechanical, olfactory, auditory) to locate a host to feed on (both plant and animal), for mating and oviposition, as well as to avoid predators at both the larval and adult stages2,3. Among these senses, olfaction plays a critical role in the above mentioned behaviors, in particular for medium to long-range detection of odorant molecules2,3. Odors emitted by a host or an oviposition site are detected by various specific olfactory receptors (e.g., GRs, ORs, IRs) located on the mosquito palps proboscis, tarsi, and antennae2,3.
As olfaction is a key component of their host-seeking (plant and animal), mating and oviposition behaviors, it thus constitutes an ideal target to study to develop new tools for mosquito control4. Research on repellents (e.g., DEET, IR3535, picaridin) and baits (e.g., BG sentinel human lure) is extremely prolific5, but because of the current challenges in mosquito control (e.g., insecticide resistance, invasive species), it is essential to develop new efficient control methods informed by the mosquito biology.
Many techniques (e.g., olfactometer, landing assays, electrophysiology) have been used to assess the bioactivity of compounds or mixtures of compounds in mosquitoes. Among them, electroantennography (or electroantennograms (EAGs)) can be used to determine whether the odorants are detected by the mosquito antennae. This technique was initially developed by Schneider6 and has been used in many different insect genera since then, including moths7,8,9, bumblebees10,11, honeybees12,13, and fruit flies14,15 to name a few. Electroantennography has also been employed using various protocols, including single or multiple antennae in mosquitoes16,17,18,19,20,21,22,23,24,25.
Mosquitoes are relatively small and delicate insects with rather thin antennae. While performing EAGs on larger insects such as moths or bumblebees is relatively easy because of their larger size and thicker antennae, conducting EAGs in mosquitoes can be challenging. In particular, maintaining a good signal-to-noise ratio and a lasting responsive preparation are two major requirements for data reproducibility and reliability.
The step-by-step guide to low noise EAGs proposed here directly offers solutions to these limitations and make this protocol applicable to several mosquito species from various genera, including Aedes, Anopheles, Culex, and Toxorhynchites, and describes the technique for both females and males. Electroantennography offers a quick yet reliable way to screen and determine bioactive compounds that can then be leveraged in bait development after valence has been determined with behavioral assays.
1. Saline solution preparation
2. Odor preparation and storage
3. Mosquito separation
4. Electrode holder and capillary preparation
5. EAG rig preparation (Figure 1)
6. Mosquito head preparation and mounting (Figure 2)
7. Recordings
8. Cleaning
9. Data analyses
Electroantennography is a powerful tool to determine whether a chemical or blend of chemicals is detected by an insect antenna. It can also be used to determine the detection threshold for a given chemical using a gradual increase of concentration (i.e., dose curve response, Figure 4B). Moreover, it is useful to test the effects of repellent on the response to host-related odors29.
Positive and negative controls should always be used in EAGs. Here, benzaldehyde was used as a positive control (Figure 3B, 3C, 4A). This compound has been found to elicit an antennal response in all mosquito species tested so far24,25,29. A negative control should also be used and can consist of the solvent used for diluting the chemicals (e.g., mineral or paraffin oils, hexane, etc.) and should not elicit a response (Figure 3B, 3C, 4A).
Indeed, when conducting EAGs, a deflection should not be noted when applying the control (Figure 3B, 3C, 4A). If a response is observed, either the syringe, the solvent control, and/or the odor line is likely contaminated. If it is the case, a new solution should be prepared, the syringe cleaned with 100% ethanol and dried and/or the airline decontaminated by rinsing with 100% ethanol and dried. If the chosen control elicits a response (e.g., ethanol), the value obtained in -mV for the control should be subtracted from the value obtained for ethanol and tested chemical combined to assess the impact of the tested chemical on the antennae.
Mosquito species vary in their ability to respond to various compounds as well as in the magnitude of their response. For example, Toxorhynchites mosquitoes produce very large EAGs in comparison to Ae. aegypti, An. Stephensi and Cx. quinquefasciatus (Figure 3C, Figure 4A).
In EAGs, the second pulse and the following usually lead to smaller EAG responses. The presentation of one odorant can also affect the response to the following, so it is important to randomize the odorant order and multiple assays to efficiently test a panel of odorants (unless a dose-response curve is performed). Moreover, separating pulses (e.g., 5 s) and odorants (e.g., 45 s) presentation will help to optimize the EAG responses.
The volatility of the tested chemicals varies and can affect the olfactory response and potentially lead to a delayed response if the tested chemical has very low volatility. Chemical volatility and solubility should be known before conducting EAGs to optimize the assay. The solvent used to prepare the dilutions should also be carefully selected (e.g., ethanol, hexane, mineral, or paraffin oil). Moreover, concentrations should be chosen wisely and should ideally be ecologically relevant. A 1% or 0.1% concentration is often used but is relatively high and not necessarily representative of what the insects can experience in nature. Yet, it is useful to screen compounds with relatively high concentrations in some cases (e.g., for bait development). Repellents can be tested at their commercially available concentration (e.g., DEET is typically sold at a 40% concentration).
If coupled with gas chromatography (i.e., GC-EADs)25, the compounds eliciting a response can be identified with a GC-MS and then tested individually at various concentrations or in mixtures with EAGs. It is worth mentioning that the valence of the tested chemicals cannot be determined with EAGs. Only a complementary behavioral experiment (e.g., olfactometer, feeding assay) can assess whether the chemical detected by the antennae is attractive, repellent, or neutral to the mosquito. Finally, EAG experiments are only showing responses of the peripheral nervous system.
Figure 1: Electroantennogram setup comprised of: A) Microscope: the microscope used should allow the experimenter to clearly see the preparation to allow the mosquito antennae tips to be inserted in the recording electrodes. B) Cold light lamp: the lamp should be turned off when the recordings begin. C) Vacuum line: this reduces the risk of accumulation of the odorants around the mosquito head preparation, which could result in antennal responses decoupled from actual stimulation. D) Micromanipulators (x2): these will allow for very fine electrode holders movements, which is required for inserting the mosquito antennae in the capillary of the recording electrode. E) Recording electrode holder. F) Reference electrode holder. G) Head stage: both electrodes are plugged in the head stage which is then connected to the amplifier. H) Main airline: a constant clean airflow bathed the mosquito head. The flow rate is regulated by a flowmeter. I) Syringe for odor delivery connected to the solenoid valve and flowmeter; J) Air table: the air table will reduce noise. K) Faraday cage: The Faraday cage will prevent electric noise. Please click here to view a larger version of this figure.
Figure 2: Step-by-step Aedes albopictus mosquito head preparation for EAG recordings. A) Female mosquito on its back on an icy plate to verify that both antennae are intact. B) Last segment of the antennae excision with micro scissors. C) Antennae are dipped in electrode gel. D) The antennae stick together after pulling them out. Only one segment of each antenna should be in the electrode gel. E) Mosquito head excision. F) Head mounted on the reference electrode. It should be stable enough to be moved to the EAG rig. A'-F'. Same steps as presented above for male EAGs. Please click here to view a larger version of this figure.
Figure 3: Schematic of the mosquito EAG and raw EAG traces. A) EAG schematic (left) and characteristics of the EAG response (right). (Left) The mosquito head is mounted between a reference electrode and a recording electrode connected to an amplifier. The antennae are bathed in a constant airflow in which odorant stimuli are pulsed. Detection of a chemical leads to a deflection (in mV) in the signal. (right) The chemical detection leads to cell depolarization (DPR) followed by cell repolarization (RPR) until return to baseline. The odorant pulse is represented by the gray rectangle. The red line indicates the amplitude of the EAG response. B) Screenshot of the WinEDR software highlighting a whole EAG recording trace of a Culex quinquefasciatus female mosquito. Top: unfiltered (i.e., raw) signal. Middle: 1 s odor pulses are indicated by numbers. Bottom: filtered (i.e., 1.5 Hz low pass) signal to 3 odorants and a control (mineral oil). Note the deflections in response to 1% 1-hexanol (1), 1% benzaldehyde (2), and 1% butyric acid (3). Note the absence of response to the negative control, mineral oil (4). C) From left to right: Representative EAG responses (in mV) to 1% benzaldehyde (top) and a mineral oil control (bottom) in females Aedes aegypti, Anopheles stephensi, Culex quinquefasciatus, and Toxorhynchites rutilus septentrionalis. The one-second pulse is represented by the colored rectangle above the EAG trace. Note the large deflection in response to benzaldehyde and the lack of response to the mineral oil. Also, note the different scale in Toxorhynchites rutilus septentrionalis. Please click here to view a larger version of this figure.
Figure 4: Example representation of EAG results and their statistical analyses. A. Average EAG responses of Culex quinquefasciatus (N = 8), Anopheles stephensi (N = 10), Aedes aegypti (N = 8) and Toxorhynchites rutilus septentrionalis (N = 7) females to 1% 1-hexanol (green), 1% butyric acid (orange), 1% benzaldehyde (yellow) and mineral oil (blue). B. Culex quinquefasciatus females EAG dose-response curve for 1-hexanol (left) (N = 9) and benzaldehyde (right) (N = 8). Bars represent standard error of the mean. Letters above error bars indicate statistical differences (Pairwise Wilcoxon rank sum test with a Bonferroni correction). Please click here to view a larger version of this figure.
Olfactory mediated behaviors are affected by many factors, including physiological (e.g., age, time of day) and environmental (e.g., temperature, relative humidity)30. Thus, when conducting EAGs, it is essential to use insects that are in the same physiological status (i.e., monitoring for age, starving, mating)31 and to also maintain a warm and humid environment around the preparation to avoid desiccation. A temperature around 25 °C is ideal and 60% to 80% humidity for the main airline. This can be easily achieved by placing a bubbler on the main airline circuit.
Moreover, it is important to consider the ecology of each species to obtain results that are relevant to the insect’s biology. For example, if using a nocturnal species, consider inversing their light cycle to test their response during their subjective night. Choosing to conduct EAGs at specific moments of the day (i.e., when the insect is active) is also important. For example, if using Ae. aegypti mosquitoes, consider doing the experiments during the peaks of activity of this species (i.e., early in the day and later afternoon). Again, the light cycle can be easily shifted for convenience using climatic chambers or light boxes with an inversed light program using a programmable timer32. Eilerts et al.33 and Krishnan et al.34, have shown that the sensitivity to specific odorants varies throughout the day. Thus, a good knowledge of the insect’s ecology and biology will guaranty more accurate results.
Noise (either electrical or mechanical) can be easily introduced in EAGs. For example, mechanical perturbations can be created by an AC system blowing air towards an EAG preparation. Electrical noise can be reduced with the Humbug, but, if persisting, can be tracked down by plugging elements and grounding them to the Faraday cage using alligator clips (Figure 3B). This applies to all the elements present around the preparation (i.e., microscope, lamp, micromanipulators). Some pieces of equipment in the Faraday cage should be unplugged before recording as they might still produce electrical noise (e.g., cold light source) or placed outside the cage. Another type of “noise” is of olfactory nature. The experimenter should avoid wearing perfume or use strongly scented shampoo or detergent. Indeed, many compounds found in these can be detected by mosquitoes (e.g., linalool, citronellol, geraniol, eugenol) and may interfere and affect the results of the experiments. Wearing a lab coat and gloves is also essential to limit unwanted contamination of the airline, syringes, and electrodes.
The presented protocol has the advantage of being easily applicable to all mosquito species, in both males and females, while extending the longevity of the preparation (> 30 min) and with limited variability between preparations. This method leads to very minimal noise in the EAG signal, which allows for testing chemicals at very low concentrations. Once the dissection and mounting steps have been mastered, this technique can produce reliable data in a relatively short amount of time, and straightforward data analyses.
Electroantennography only allows the experimenter to assess whether the mosquito can detect a chemical or not. However, to determine the valence of this chemical, complementary behavioral assays, such as olfactometer assays, are critical to determining whether a specific odorant or mixture is attractive, repellent, or neutral in order to develop efficient tools for mosquito control35.
The authors have nothing to disclose.
I am grateful to Dr. Clément Vinauger and Dr. Jeffrey Riffell for helpful discussions. The following reagents were obtained through BEI Resources, NIAID, NIH: Anopheles stephensi, Strain STE2, MRA-128, contributed by Mark Q. Benedict; Aedes aegypti, Strain ROCK, MRA-734, contributed by David W. Severson; Culex quinquefasciatus, Strain JHB, Eggs, NR-43025. The author thanks Dr. Jake Tu, Dr. Nisha Duggal, Dr. James Weger and Jeffrey Marano for providing Culex quinquefasciatus and Anopheles stephensi (strain: Liston) mosquito eggs. Aedes albopictus and Toxorhynchites rutilus septentrionalis are derived from field mosquitoes collected by the author in the New River Valley area (VA, USA). This work was supported by The Department of Biochemistry and The Fralin Life Science Institute.
Air table Clean Bench | TMC | https://www.techmfg.com/products/labtables/cleanbench63series/accessoriess | Noise reducer |
Analog-to-digital board | National Instruments | BNC-2090A | |
Benchtop Flowbuddy Complete | Genesee Scientific | 59-122BC | To anesthesize mosquitoes |
Borosillicate glass capillary | Sutter Instrument | B100-78-10 | To make the recording and references capillaries |
Chemicals | Sigma Aldrich | Benzaldehyde: 418099-100 mL; Butyric acid: B103500-100mL; 1-Hexanol: 471402-100mL; Mineral oil: M8410-1L | Chemicals used for the experiments presented here |
CO2 | Airgas or Praxair | N/A | To anesthesize mosquitoes |
Cold Light Source | Volpi | NCL-150 | |
Disposable syringes | BD | 1 mL (309628) / 3 mL (309657) | |
Electrode cables | World Precision Instruments | 5371 | |
Electrode gel salt free | Parkerlabs | 12-08-Spectra-360 | |
Faraday cage | TMC | https://www.techmfg.com/products/electric-and-magnetic-field-cancellation/faradaycages | Noise reducer |
Flowmeters | Bel-art | 65 mm (H40406-0010) / 150 mm (H40407-0075) | One of each |
GCMS vials and caps | Thermo-fisher scientific | 2-SVWKA8-CPK | To prepare odorant dilutions |
Glass syringes (Fortuna) | Sigma Aldrich | Z314307 | For odor delivery to the EAG prep |
Humbug | Quest Scientific | http://www.quest-sci.com/ | Noise reducer |
2 mm Jack Holder, Narrow, 90 deg., With Wire | A-M Systems | 675748 | Electrode holder |
Magnetic bases | Kanetec | MB-FX | x 2 |
MATLAB + Toolboxes | Mathworks | https://www.mathworks.com/products/matlab.html | For delivering the pulses |
Medical air | Airgas or Praxair | N/A | For main airline |
Microscope | Nikkon | SMZ-800N | |
Micromanipulators Three-Axis Coarse/Fine Compact Micromanipulator | Narishige | MHW-3 | x 2 |
Microelectrode amplifier with headstage | A-M Systems | Model 1800 | |
Mosquito rearing supplies | Bioquip | https://www.bioquip.com/Search/WebCatalog.asp | |
Needles | BD | 25G (305127) / 21G (305165) | |
Pasteur pipettes | Fisher Scientific | 13-678-6A | For odor delivery to the EAG prep |
PTFE Tubing of different diameters | Mc Master Carr | N/A | To connect solenoid valve, flowmeter, airline ect. |
30V/5A DC Power Supply | Dr. Meter | PS-305DM | |
R version 3.5.1 | R project | https://www.r-project.org/ | For data analyses |
Relay for solenoid valve | N/A | Custom made | |
Silver wire 0.01” | A-M Systems | 782500 | |
Solenoid valve (3-way) | The Lee Company | LHDA0533115H | |
WinEDR software | Strathclyde Electrophysiology Software | WinEDR V3.9.1 | For EAG recording |
Whatman paper | Cole Parmer | UX-06648-03 | To load chemical in glass syringe / Pasteur pipette |